Proteins are the machines of life and the targets of essentially all pharmaceuticals. One of the most important properties of proteins is their ability to interact with other proteins. While numerous protein-protein interactions are critical to the function of normal healthy cells, other protein-protein interactions are associated with infectious diseases, inheritable diseases, and cancer. Identifying these disease-associated protein-protein interactions is central to the discovery of new therapeutics. Once a disease-associated protein-protein interaction has been identified, researchers can begin the process of developing a therapeutic molecule that is capable of disrupting the detrimental protein-protein interaction. This effort to develop a therapeutic molecule must be guided by an assay that allows the researchers to rapidly and effectively test candidate therapeutic molecules for their ability to disrupt the specific protein-protein interaction of interest (Michnick et al., 2007).
For both the discovery of previously unknown protein-protein interactions, as well as assays of known protein-protein interactions, cell-based assays in which the interaction of interest is associated with a change in the visible fluorescence of the cells are particularly powerful.
Fluorescence is a well understood phenomenon in which the absorbance of higher energy (more blue shifted) light by a molecular species leads to the emission of lower energy (more red shifted) light with a very short time delay (typically nanoseconds). Fluorescence is the preferred readout for cell-based assays because it is extremely sensitive, versatile, and can be implemented in minimally invasive ways.
The two main challenges of using a fluorescent cell-based assay are: 1) introducing the fluorescent molecule into a cell; and 2) making the change in fluorescence intensity or color meaningfully correlated with the protein-protein interaction or other biochemical event of interest. The first of these two challenges is most effectively addressed by using fluorescent protein (FP) technology. FPs are naturally occurring proteins that have been found in various marine organisms from phyla Cnidaria (i.e., Hydrozoan jellyfish and Anthozoan coral) (Shimomura et al., 1962; Matz et al., 1999), Chordata (i.e., lancelet) (Deheyn et al., 2007; Shaner et al., 2013), and Arthropoda (i.e., a copepod crustacean) (Masuda et al., 2006). The corresponding genes encoding these proteins have been cloned from their host organisms or resynthesized in the lab, and then extensively engineered in the laboratory to produce improved FPs for research applications in biological imaging (Campbell and Davidson, 2010). Available methods to address the second challenge, and use FPs for detecting protein-protein interactions or other biochemical processes of interest, are also known.
Methods for Detecting Protein-Protein Interactions Using Fluorescent Proteins
While the strategies for using fluorescent proteins (FPs) as markers of gene expression, protein localization, and organelle structure are well-established, current methods for converting FPs into active indicators of protein-protein interactions and biochemistry in live cells remain few in number. The two standard methods for detecting protein-protein interactions in live cells are: 1) Interaction-induced reassembly of an FP that has been genetically split into two fragments (Ghosh et al., 2000; Hu et al., 2002; Alford et al., 2012; Nyfeler et al., 2005; Kerppola, 2008); and 2) Förster resonance energy transfer (FRET) between two different hues of FP (Miyawaki et al., 1997; Xu et al., 1998). For more than a decade, both of these methods have been exploited in a variety of applications that have led to numerous important biological insights. However, taken as a group, these methods suffer from a few shortcomings. For example, FRET-based biosensors tend to have relatively small fluorescent responses and are challenging to implement with multiple fluorescent probes (Carlson and Campbell, 2009); and the slow and irreversible nature of split FP complementation means that it cannot be used to visualize reversible protein-protein interactions and may also suffer from artifacts due to the capturing of weak or transient interactions (Kodama and Hu, 2012).
Dimerization-dependent fluorescent protein (ddFP) technology was recently introduced as a versatile method that attempted to address some of the drawbacks associated with split FP reconstitution and FRET assays, while providing new opportunities for the construction of biosensors (Alford et al., 2012; Alford et al., 2012). A ddFP is a pair of quenched or non-fluorescent FP monomers that can associate to form a fluorescent heterodimer. One of the monomers (“copy A” or “fluorogenic monomer”) contains a fully formed chromophore that is quenched in the monomeric state. The second monomer (“copy B” or “dark monomer”) does not form a chromophore itself and only acts to substantially increase the fluorescence of copy A upon formation of the AB heterodimer. In the green and red fluorescent versions of ddFP, the A copies are referred to as GA and RA, respectively. For both GA and RA, a corresponding B copy (i.e., GB and RB) was engineered that had been optimized with respect to formation of its respective fluorogenic heterodimer. DdFPs have been used individually as intensiometric biosensors for a variety of biochemical processes including protein-protein interaction, protease activity, and membrane-membrane proximity. (Alford et al., 2012; Alford et al., 2012). One example of a commonly used protease assay is the monitoring of caspase activity during the process of apoptosis (programmed cell death). To make indicators of protease activity, proteins were expressed as a tandem genetically fused AB heterodimer with a linker that contains a protease substrate. For example, caspase-3 activity indicators were created based on a linker containing the substrate sequence Asp-Glu-Val-Asp (DEVD, SEQ ID NO:1) (Xu et al., 1998) and green, red and yellow ddFPs (Alford et al., 2012; Alford et al., 2012). Traditionally, caspase-3 biosensors have relied on the loss of FRET that occurs when the substrate sequence linking a donor FP to an acceptor FP is cleaved by the protease of interest (Xu et al., 1998; Ai et al., 2008). One disadvantage of ddFPs relative to FRET for detecting protein-protein interactions or protein cleavage due to protease activity is that ddFPs provide an intensiometric (i.e., single color increase or decrease) fluorescence response, while FRET provides a ratiometric (i.e., color change) response. Generally speaking, ratiometric changes are more amenable to quantitative analysis.
U.S. Pat. No. 7,666,606, “Protein-protein interaction detection system using fluorescent protein microdomains” describes the use of a ‘microdomain’, or a peptide portion of the fluorescent protein. Other patents describing fluorescent technology include U.S. Pat. No. 7,166,424, “Fragments of fluorescent proteins for protein fragment complementation assays”; U.S. Pat. No. 8,426,153, “Linked peptides fluorogenic biosensors”; U.S. Pat. No. 6,294,330, “Protein fragment complementation assays for the detection of biological or drug interactions”; U.S. Pat. No. 6,828,099, “Protein-fragment complementation assays (PCA) for the detection of protein-protein, protein-small molecule and protein-nucleic acid interactions based on the E. Coli TEM-1 beta-lactamase.”; and U.S. Pat. No. 6,897,017, “In vivo library versus library selection of optimized protein-protein interactions”.
There is a need for a novel method of analyzing protein-protein interactions for facilitating high throughput assaying of drug targets.
This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention.